【摘要】：Crizotinib是靶向MET、ALK融合基因和ROS1的多靶點(diǎn)受體酪氨酸激酶抑制劑。Crizotinib作為MET抑制劑被研發(fā),但首先被FDA批準(zhǔn)用于EML4-ALK融合基因陽性的局部進(jìn)展期及轉(zhuǎn)移性肺腺癌的一線治療,并同時(shí)建議用于存在MET基因擴(kuò)增的肺癌患者。然而同大多數(shù)分子靶向治療藥物類似,半數(shù)以上患者在初始治療1年內(nèi)發(fā)生耐藥,其原因包括原發(fā)性及獲得性耐藥突變,多種旁路癌基因的激活等。作為細(xì)胞對外界刺激的一種應(yīng)激性存活機(jī)制,自噬參與了多種腫瘤細(xì)胞的耐藥。常見的抗腫瘤治療如化療、放療、生物靶向治療等均可以引起腫瘤細(xì)胞自噬。Crizotinib能否誘導(dǎo)肺癌細(xì)胞自噬發(fā)生,從而導(dǎo)致其耐藥尚不清楚。目的研究Crizotinib對肺癌細(xì)胞自噬水平的影響及自噬在肺癌細(xì)胞耐藥中所起的作用；分析Crizotinib誘導(dǎo)自噬發(fā)生的具體分子機(jī)制；在此基礎(chǔ)上觀察抑制自噬是否增加肺癌細(xì)胞對Crizotinib的敏感性。方法通過Western blot.免疫熒光、透射電鏡等方法觀察Crizotinib處理后肺癌細(xì)胞SPC-A1、A549、H2228中自噬相關(guān)蛋白表達(dá)情況及自噬體的形成；Western blot分析Criztinib處理后肺癌細(xì)胞MET, STAT3, AKT/MTOR, ERK等信號通路關(guān)鍵分子的表達(dá)情況；采用MTS方法檢測通過自噬抑制劑或下調(diào)自噬關(guān)鍵基因Beclinl抑制自噬對Crizotinib抗腫瘤作用的影響；采用流式細(xì)胞儀定量分析藥物處理后細(xì)胞凋亡比率；建立肺癌裸鼠移植瘤模型評估自噬抑制劑對Crizotinib的增敏作用。結(jié)果Crizotinib能夠抑制多種肺癌細(xì)胞株的活力,用Crizotinib處理表達(dá)MET的SPC-A1細(xì)胞,表達(dá)EML4-ALK的H2228細(xì)胞,和同時(shí)具有MET表達(dá)和KRAS突變的A549細(xì)胞,均可見細(xì)胞自噬水平的上升,表現(xiàn)為LC3 Ⅰ向LC3 Ⅱ轉(zhuǎn)換增多；免疫熒光顯示LC3Ⅱ puncta增多及電鏡下自噬體的增加等。Crizotinib能夠抑制MET及其下游STAT3、AKTYMTOR和ERK的磷酸化水平。進(jìn)一步的研究發(fā)現(xiàn)Crizotinib誘導(dǎo)肺癌細(xì)胞自噬主要是通過分階段的抑制胞質(zhì)及胞核中的STAT3信號通路實(shí)現(xiàn),胞質(zhì)中STAT3的抑制導(dǎo)致EIF2AK2與其解離,繼而磷酸化EIF2A而誘導(dǎo)自噬增加,而胞核中STAT3的抑制導(dǎo)致其轉(zhuǎn)錄激活的BCL2水平下降,進(jìn)而促進(jìn)自噬水平的持續(xù)增加。體外研究表明使用自噬抑制劑或下調(diào)自噬關(guān)鍵基因Beclinl抑制自噬促進(jìn)Crizotinib對肺癌細(xì)胞的增殖抑制作用。不同濃度的Crizotinib聯(lián)合CQ或3-MA作用于SPC-A1和A549細(xì)胞后對其抑制作用顯著高于Crizotinib單藥組,(p0.001)。以shRNA Beclinl轉(zhuǎn)染SPC-A1和A549細(xì)胞,也能夠顯著提高其對Crizotinib的敏感性。流式細(xì)胞儀檢測結(jié)果表明與Crizotinib單藥處理相比,聯(lián)合CQ和3-MA時(shí),SPC-A1細(xì)胞凋亡率顯著增加(p0.05)。裸鼠移植瘤模型表明自噬抑制劑HCQ增加Crizotinib對SPC-A1細(xì)胞移植瘤生長的抑制作用。Crizotinib聯(lián)合HCQ組抑制腫瘤更為明顯,而不顯著增加毒性。結(jié)論多靶點(diǎn)酪氨酸激酶抑制劑Crizotinib能夠誘導(dǎo)肺癌細(xì)胞保護(hù)性自噬,其機(jī)制是通過階段性的抑制胞漿和胞核內(nèi)的STAT3信號通路實(shí)現(xiàn)。抑制自噬促進(jìn)Crizotinib在體內(nèi)外對肺癌細(xì)胞的增殖抑制作用,提高細(xì)胞凋亡率。聯(lián)合自噬抑制劑可以作為增敏Crizotinib治療肺癌的一種新的策略。
[Abstract]:Crizotinib is a multi-target receptor tyrosine kinase inhibitor targeting MET, ALK fusion genes, and ROS1. Crizotinib is developed as a MET inhibitor, but is first approved by the FDA for first-line treatment for EML4-ALK fusion gene-positive local progression and metastatic lung adenocarcinoma, and is also recommended for patients with lung cancer with MET gene amplification. However, similar to most of the molecular targeted therapies, more than half of the patients experienced resistance in the initial treatment for 1 year, including primary and acquired resistance mutations, activation of multiple by-pass oncogene, and the like. As a stress-based survival mechanism for external stimulation of cells, autophagy is involved in the resistance of multiple tumor cells. Common anti-tumor treatments such as chemotherapy, radiotherapy, and biological targeted therapy can cause autophagy of the tumor cells. The ability of rizotinib to induce autophagy in lung cancer cells leads to a lack of clarity in its drug resistance. Objective To study the effect of crizotinib on autophagy of lung cancer cells and the role of autophagy in the drug resistance of lung cancer cells, and to analyze the specific molecular mechanism of the induction of autophagy by Crizotinib. Methods Western blot was used. The expression of autophagy in lung cancer cells SPC-A1, A549 and H2228 and the formation of self-phagocytosis were observed by immunofluorescence and transmission electron microscopy. The expression of MET, STAT3, AKT/ MOR, ERK and other signal pathways in lung cancer cells after treatment with criztepotinib was analyzed by Western blot. The effect of autophagy on the anti-tumor effect of Crizotinib by autophagy inhibitor or down-regulated autophagy is detected by MTS method, and the cell apoptosis ratio after drug treatment is quantitatively analyzed by flow cytometry; and the sensitization effect of the autophagy inhibitor on the Critzotinib is evaluated by establishing a nude mouse transplantation tumor model of the lung cancer. Results The activity of Critzotinib could inhibit the activity of multiple lung cancer cell lines. The SPC-A1 cells expressing MET were treated with Crizotinib, and the H2228 cells expressing EML4-ALK and A549 cells with MET expression and KRAS mutation were observed. Immunofluorescence showed the increase of LC3 鈪，

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